Substitution mutant receptors and their use in an ecdysone receptor-based inducible gene expression system

Abstract

This invention relates to the field of biotechnology or genetic engineering. Specifically, this invention relates to the field of gene expression. More specifically, this invention relates to novel nuclear receptors comprising a substitution mutation and their use in a nuclear receptor-based inducible gene expression system and methods of modulating the expression of a gene within a host cell using this inducible gene expression system.

We claim: 
     
       1. A method of modulating the expression of a gene in a host cell comprising the steps of:
 (a) introducing into the host cell a gene expression modulation system; comprising:
 (i) a first gene expression cassette comprising a polynucleotide that encodes a first polypeptide, comprising:
 (1) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and 
 (2) a first nuclear receptor ligand binding domain; and 
 
 (ii) a second gene expression cassette comprising a polynucleotide that encodes a second polypeptide, comprising:
 (1) a transactivation domain; and 
 (2) a second nuclear receptor ligand binding domain, 
 
 wherein one of the first and second nuclear receptor ligand binding domains is a Group B nuclear receptor ligand binding domain comprising a substitution mutation at a position selected from the group consisting of
 (1) 401 or 429 of SEQ ID NO:1, 
 (2) 401 and 429 of SEQ ID NO:1, 
 (3) 337, 462, 470, or 473 of SEQ ID NO:2, 
 (4) 450, 451, and 452 of SEQ ID NO:2, 
 (5) 455, 456, 457, and 458 of SEQ ID NO:2, 
 (6) 475, 476, 477, 478, and 479 of SEQ ID NO:2, and 
 (7) 481, 482, and 483 of SEQ ID NO:2; and 
 
 the other of said nuclear receptor ligand binding domains is a nuclear receptor ligand binding domain that dimerizes with said Group B nuclear receptor ligand binding domain, and 
 
 (b) introducing into the host cell a ligand; wherein the gene to be modulated is a component of a gene expression cassette comprising:
 (i) a response element recognized by the DNA binding domain; 
 (ii) a promoter that is activated by the transactivation domain; and 
 (iii) a gene whose expression is to be modulated; 
 
 whereby upon introduction of the ligand into the host cell, expression of the gene of (b)(iii) is modulated. 
 
     
     
       2. The method according to  claim 1 , wherein the ligand is
 a) a compound of the formula: 
 
       
         
           
           
               
               
           
         
         wherein: 
         E is a (C 4 -C 6 )alkyl containing a tertiary carbon or a cyano(C 3 -C 5 )alkyl containing a tertiary carbon; 
         R 1  is H, Me, Et, i-Pr, F, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, SCN, or SCHF 2 ; 
         R 2  is H, Me, Et, n-Pr, i-Pr, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe 2 , NEt 2 , SMe, SEt, SOCF 3 , OCF 2 CF 2 H, COEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, OCF 3 , OCHF 2 , O-i-Pr, SCN, SCHF 2 , SOMe, NH—CN, or joined with R 3  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon; 
         R 3  is H, Et, or joined with R 2  and the phenyl carbons to which R 2  and R 3  are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon; 
         R 4 , R 5  and R 6  are independently H, Me, Et, F, Cl, Br, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or Set; 
         b) an ecdysone, 20-hydroxyecdysone, ponasterone A, or muristerone A; 
         c) an oxysterol, a 22(R) hydroxycholesterol, 24(S) hydroxycholesterol 25-epoxycholesterol, 70901317, 5-alpha-6-alpha-epoxycholesterol-3-sulfate, 7-ketocholesterol-3-sulfate, farnesol, a bile acid, a 1,1-biphosphonate este a Juvenile hormone III; or 
         d) a 9-cis-retinoic acid, 4-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-napthyl)-ethenyl) benzoic acid (3-methyl-TTEB), ((E)-2)2-(5,6,7,8-tetrat-hydro-3,5,5,8,8-pentamethyl-2-napthyl)propen-1-yl)-4-thiophenecarboxylic acid), 2-(5,6,7,8-tetra-hydro-3,5,5,8,8-tetramethyl-2-naphthyl)-2-(carboxyphenyl)-1,3-dioxolane, 4-(5H-2,3-(2,5-dimethyl-2,5-hexano)-5-methyl-dibenzo (b,e) (1.4)diazepin-11-yl)-benzoic acid (HX600) or thiadiazepin analogs thereof, 3,7,11,15-tetramethyl hexadecanoic acid (phytanic acid), 6-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl) cyclopropyl) nicotinic acid, 2-(4-carboxyphenyl)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1,3-dithiane, or 4-(2-methyl)-1-(5,6,7,8-tetrahydro-5,5,8-tetramethyl-2-napthalenyl)propenyl)benzoic acid. 
       
     
     
       3. The method according to  claim 1 , wherein the method further comprises introducing into the host cell a second ligand, wherein the second ligand is 9-cis-retinoic acid or a synthetic analog of a retinoic acid.